Absolute Reticulocyte Count Acts as a Surrogate for Fetal Hemoglobin in Infants and Children with Sickle Cell Anemia

Hemoglobin switching is largely complete in humans by six months of age. Among infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). The objective of this study was to determine if absolute reticulocyte count (ARC) is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study. Hematologic data, including ARC and HbF levels, were measured at steady state. F-cells were enumerated by flow cytometry. Initial studies compared infants with ARC greater than or equal to 200 K/μL (ARC ≥ 200) based upon the previously reported utility of this threshold as a predictive marker for SCA severity. Mean HbF and F-cell levels were significantly lower in the ARC ≥ 200 group when compared to the ARC < 200 group. Both HbF and F-cell percentages were negatively correlated to ARC in infants and in children between the ages of 1 and 9 years. However, the inverse relationship was lost after the age of 10 years. Overall, decreased expression and distribution of HbF during childhood SCA is well-correlated with increased reticulocyte production and release into the peripheral blood. As such, these data further support the clinical use of reticulocyte enumeration as a disease severity biomarker for childhood sickle cell anemia.     

HMGB1 Mediates Anemia of Inflammation in Murine Sepsis Survivors

Patients surviving sepsis develop anemia, but the molecular mechanism is unknown. Here we observed that mice surviving polymicrobial gram-negative sepsis develop hypochromic, microcytic anemia with reticulocytosis. The bone marrow of sepsis survivors accumulates polychromatophilic and orthochromatic erythroblasts. Compensatory extramedullary erythropoiesis in the spleen is defective during terminal differentiation. Circulating tumor necrosis factor (TNF) and interleukin (IL)-6 are elevated for 5 d after the onset of sepsis, and serum high-mobility group box 1 (HMGB1) levels are increased from d 7 until at least d 28. Administration of recombinant HMGB1 to healthy mice mediates anemia with extramedullary erythropoiesis and significantly elevated reticulocyte counts. Moreover, administration of anti-HMGB1 monoclonal antibodies after sepsis significantly ameliorates the development of anemia (hematocrit 48.5 ± 9.0% versus 37.4 ± 6.1%, p < 0.01; hemoglobin 14.0 ± 1.7 versus 11.7 ± 1.2 g/dL, p < 0.01). Together, these results indicate that HMGB1 mediates anemia by interfering with erythropoiesis, suggesting a potential therapeutic strategy for anemia in sepsis.     

Effect of insulin-induced hypoglycemia on the serum concentrations of thyroxine,triiodothyronine and reverse triiodothyronine

The effect of insulin-induced hypoglycemia on serum thyroid hormone concentrations was studied in nine healthy individuals. Before, during and after the hypoglycemia blood samples were taken for measurement of the concentrations of glucose, thyroxine (T₄), triiodothyronine (T₃), reverse triiodothyronine (rT₃), catecholamines and pituitary hormones.There was no change in the mean serum T₄ level (± the standard error of the mean) of 67 ± 2 μg/l. However, the T₃ concentrations rose from a mean basal level of 1.86 ± 0.06 μg/l to a mean peak of 2.51 ± 0.21 μg/l (P < 0.01) at 45 minutes after the insulin injection, and the rT₃ concentrations fell from a mean basal level of 0.184 ± 0.008 μg/l to a mean nadir of 0.171 ± 0.022 μg/l (not a significant change). The mean peak epinephrine level was 545 ± 103 ng/l and it occurred between 30 and 45 minutes after the insulin injection; the mean peak norepinephrine level was 584 ± 114 ng/l and it occurred between 30 and 90 minutes after the injection. The growth hormone levels reached a mean peak of 26.1 ± 4.8 μg/l and the plasma cortisol levels rose to 215 ± 9 μg/l. The mean basal prolactin level was 8.5 ± 0.9 μg/l; in five subjects there was a rise to a mean peak of 50.6 ± 14.6 μg/l, whereas in the remaining four no significant increase occurred. No correlation was found between the changes in the serum T₃ concentration and any of the other factors studied.It was concluded that acute hypoglycemia is associated with a rapid increase in the serum T₃ concentration.     

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca²⁺]_i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca²⁺]_i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.     

Functional Assessment of Cardiac Responses of Adult Zebrafish (Danio rerio) to Acute and Chronic Temperature Change Using High-Resolution Echocardiography

The zebrafish (Danio rerio) is an important organism as a model for understanding vertebrate cardiovascular development. However, little is known about adult ZF cardiac function and how contractile function changes to cope with fluctuations in ambient temperature. The goals of this study were to: 1) determine if high resolution echocardiography (HRE) in the presence of reduced cardiodepressant anesthetics could be used to accurately investigate the structural and functional properties of the ZF heart and 2) if the effect of ambient temperature changes both acutely and chronically could be determined non-invasively using HRE in vivo. Heart rate (HR) appears to be the critical factor in modifying cardiac output (CO) with ambient temperature fluctuation as it increases from 78 ± 5.9 bpm at 18°C to 162 ± 9.7 bpm at 28°C regardless of acclimation state (cold acclimated CA– 18°C; warm acclimated WA– 28°C). Stroke volume (SV) is highest when the ambient temperature matches the acclimation temperature, though this difference did not constitute a significant effect (CA 1.17 ± 0.15 μL at 18°C vs 1.06 ± 0.14 μl at 28°C; WA 1.10 ± 0.13 μL at 18°C vs 1.12 ± 0.12 μl at 28°C). The isovolumetric contraction time (IVCT) was significantly shorter in CA fish at 18°C. The CA group showed improved systolic function at 18°C in comparison to the WA group with significant increases in both ejection fraction and fractional shortening and decreases in IVCT. The decreased early peak (E) velocity and early peak velocity / atrial peak velocity (E/A) ratio in the CA group are likely associated with increased reliance on atrial contraction for ventricular filling.     

In vivo CD8+ T Cell Dynamics in the Liver of Plasmodium yoelii Immunized and Infected Mice

Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in situ, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS_280–288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.     

Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes

Cell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117) undergoing IVF/Intra-cytoplasmic sperm injection (ICSI) procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome) than from patients with normal ovarian reserve (2.7 ± 2.7 ng/μl versus 1.7 ± 2.3 ng/μl, respectively, p = 0.03). Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days) or high total dose of gonadotropins (≥ 3000 IU/l) than in women who received short stimulation duration (7–10 days) or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/μl versus 1.5 ± 1.9 ng/μl, p = 0.008; 2.2 ± 2.3 ng/μl versus 1.5 ± 2.1 ng/μl, p = 0.01, respectively). Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03). In multivariate analysis, the Receiving Operator Curve (ROC) analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66–0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management.     

Pharmacokinetics and Pharmacodynamics of Recombinant Human EPO-Fc Fusion Protein In Vivo

In this study, the in vivo pharmacokinetics and pharmacodynamics of a novel recombinant human erythropoietin (rhEPO) Fc fusion protein, rhEPO-Fc, were studied in both rodents and rhesus monkeys. Animal models of anemia induced by irradiation, cyclophosphamide and partial renal ablation were used to evaluate therapeutic effects of rhEPO-Fc. We have demonstrated that serum half-life of rhEPO-Fc was 29.5 to 38.9 h at doses of 8, 25, 80 µg/kg in rhesus monkeys and 35.5 to 43.5 h at doses of 16, 50, 160 µg/kg in rats. In anemia animal models, rhEPO-Fc dose-dependently (7.5–30.0 µg/kg in mice, 5.4–21.4 µg/kg in rats and 5.0–10.0 µg/kg in rhesus monkeys) increased reticulocyte level, followed by an increase of RBC count, hemoglobin and hematocrit levels. At reduced intervention frequency of weekly treatments, rhEPO-Fc showed similar hematopoietic effects as compared with rhEPO given three times a week. These results indicated that rhEPO-Fc could potentially be used in treatment of anemia and warrants future clinical trials.     

Percutaneous Mitral Valve Repair in Mitral Regurgitation Reduces Cell-Free Hemoglobin and Improves Endothelial Function

Background and Objective

Endothelial dysfunction is predictive for cardiovascular events and may be caused by decreased bioavailability of nitric oxide (NO). NO is scavenged by cell-free hemoglobin with reduction of bioavailable NO up to 70% subsequently deteriorating vascular function. While patients with mitral regurgitation (MR) suffer from an impaired prognosis, mechanisms relating to coexistent vascular dysfunctions have not been described yet. Therapy of MR using a percutaneous mitral valve repair (PMVR) approach has been shown to lead to significant clinical benefits. We here sought to investigate the role of endothelial function in MR and the potential impact of PMVR.

Methods and Results

Twenty-seven patients with moderate-to-severe MR treated with the MitraClip® device were enrolled in an open-label single-center observational study. Patients underwent clinical assessment, conventional echocardiography, and determination of endothelial function by measuring flow-mediated dilation (FMD) of the brachial artery using high-resolution ultrasound at baseline and at 3-month follow-up. Patients with MR demonstrated decompartmentalized hemoglobin and reduced endothelial function (cell-free plasma hemoglobin in heme 28.9±3.8 μM, FMD 3.9±0.9%). Three months post-procedure, PMVR improved ejection fraction (from 41±3% to 46±3%, p = 0.03) and NYHA functional class (from 3.0±0.1 to 1.9±1.7, p<0.001). PMVR was associated with a decrease in cell free plasma hemoglobin (22.3±2.4 μM, p = 0.02) and improved endothelial functions (FMD 4.8±1.0%, p<0.0001).

Conclusion

We demonstrate here that plasma from patients with MR contains significant amounts of cell-free hemoglobin, which is accompanied by endothelial dysfunction. PMVR therapy is associated with an improved hemoglobin decompartmentalization and vascular function.     

Circulating NOS3 Modulates Left Ventricular Remodeling following Reperfused Myocardial Infarction

Purpose

Nitric oxide (NO) is constitutively produced and released from the endothelium and several blood cell types by the isoform 3 of the NO synthase (NOS3). We have shown that NO protects against myocardial ischemia/reperfusion (I/R) injury and that depletion of circulating NOS3 increases within 24h of ischemia/reperfusion the size of myocardial infarction (MI) in chimeric mice devoid of circulating NOS3. In the current study we hypothesized that circulating NOS3 also affects remodeling of the left ventricle following reperfused MI.

Methods

To analyze the role of circulating NOS3 we transplanted bone marrow of NOS3^−/− and wild type (WT) mice into WT mice, producing chimerae expressing NOS3 only in vascular endothelium (BC−/EC+) or in both, blood cells and vascular endothelium (BC+/EC+). Both groups underwent 60 min of coronary occlusion in a closed-chest model of reperfused MI. During the 3 weeks post MI, structural and functional LV remodeling was serially assessed (24h, 4d, 1w, 2w and 3w) by echocardiography. At 72 hours post MI, gene expression of several extracellular matrix (ECM) modifying molecules was determined by quantitative RT-PCR analysis. At 3 weeks post MI, hemodynamics were obtained by pressure catheter, scar size and collagen content were quantified post mortem by Gomori’s One-step trichrome staining.

Results

Three weeks post MI, LV end-systolic (53.2±5.9μl;***p≤0.001;n = 5) and end-diastolic volumes (82.7±5.6μl;*p<0.05;n = 5) were significantly increased in BC−/EC+, along with decreased LV developed pressure (67.5±1.8mmHg;n = 18;***p≤0.001) and increased scar size/left ventricle (19.5±1.5%;n = 13;**p≤0.01) compared to BC+/EC+ (ESV:35.6±2.2μl; EDV:69.1±2.6μl n = 8; LVDP:83.2±3.2mmHg;n = 24;scar size/LV13.8±0.7%;n = 16). Myocardial scar of BC−/EC+ was characterized by increased total collagen content (20.2±0.8%;n = 13;***p≤0.001) compared to BC+/EC+ (15.9±0.5;n = 16), and increased collagen type I and III subtypes.

Conclusion

Circulating NOS3 ameliorates maladaptive left ventricular remodeling following reperfused myocardial infarction.     

Correlation of exhaled breath temperature with bronchial blood flow in asthma

In asthma elevated rates of exhaled breath temperature changes (Δe°T) and bronchial blood flow (Q_aw) may be due to increased vascularity of the airway mucosa as a result of inflammation.We investigated the relationship of Δe°T with Q_aw and airway inflammation as assessed by exhaled nitric oxide (NO). We also studied the anti-inflammatory and vasoactive effects of inhaled corticosteroid and β₂-agonist.Δe°T was confirmed to be elevated (7.27 ± 0.6 Δ°C/s) in 19 asthmatic subjects (mean age ± SEM, 40 ± 6 yr; 6 male, FEV₁ 74 ± 6 % predicted) compared to 16 normal volunteers (4.23 ± 0.41 Δ°C/s, p < 0.01) (30 ± 2 yr) and was significantly increased after salbutamol inhalation in normal subjects (7.8 ± 0.6 Δ°C/ s, p < 0.05) but not in asthmatic patients. Q_aw, measured using an acetylene dilution method was also elevated in patients with asthma compared to normal subjects (49.47 ± 2.06 and 31.56 ± 1.6 μl/ml/min p < 0.01) and correlated with exhaled NO (r = 0.57, p < 0.05) and Δe°T (r = 0.525, p < 0.05). In asthma patients, Q_aw was reduced 30 minutes after the inhalation of budesonide 400 μg (21.0 ± 2.3 μl/ml/min, p < 0.05) but was not affected by salbutamol.Δe°T correlates with Q_aw and exhaled NO in asthmatic patients and therefore may reflect airway inflammation, as confirmed by the rapid response to steroids.     

CD4 Cell Count Trends after Commencement of Antiretroviral Therapy among HIV-Infected Patients in Tigray,Northern Ethiopia: A Retrospective Cross-Sectional Study

Background

The rate and extent of CD4 cell recovery varies widely among HIV-infected patients with different baseline CD4 cell count strata. The objective of the study was to assess trends in CD4 cell counts in HIV-infected patients after initiation of antiretroviral therapy in Tigray, Northern Ethiopia.

Methods

A retrospective cross-sectional study was conducted by reviewing medical records of HIV patients who received antiretroviral treatment at twenty health centers in Tigray region during 2008–2012. Multi-stage cluster sampling technique was employed to collect data, and the data were analyzed using SPSS version 20.0 software.

Results

The median change from baseline to the most recent CD4 cell count was +292 cells/μl. By 5 years, the overall median (inter-quartile range, IQR) CD4 cell count was 444(263-557) cells/μl while the median (IQR) CD4 cell count was 342(246-580) cells/μl among patients with baseline CD4 cell counts ≤200 cells/μl, 500(241-557) cells/μl among those with baseline CD4 cell counts of 201–350 cells/μl, and 652(537-767) cells/μl among those with baseline CD4 cell counts >350 cells/μl. Higher baseline CD4 cell counts and being male were independently associated with the risk of immunological non-response at 12 months. Furthermore, it was also investigated that these factors were significant predictors of subsequent CD4 cell recovery.

Conclusions

Patients with higher baseline CD4 cell stratum returned to normal CD4 Cell counts though they had an increased risk of immunological non-response at 12 months compared to those with the least baseline CD4 cell stratum. The findings suggest that consideration be given to initiation of HAART at a CD4 cell count >350 cells/μl to achieve better immune recovery, and to HIV-infected male patients to improve their health seeking behavior.     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.     

Photosynthetic electron transport in isolated maize bundle sheath cells

Fragments of bundle sheath strands, free of mesophyll cells and showing a chlorophyll a/b ratio of 6.0 to 6.6 were prepared from Zea mays by a mechanical method. They were unable to photoreduce ferricyanide but were able to photoreduce the membrane-permeant 2,5-dimethylquinone at a rate of 250 to 420 microequivalents per hour per mg chlorophyll (μeq/hr · mg Chl) at 21 C. In the presence of the catalase inhibitor KCN, methylviologen catalyzed a Mehler reaction at a rate of 120 to 180 μeq/hr · mg Chl. This was increased to 200 to 350 μeq/hr · mg Chl when the uncoupler methylamine was added. The rate of endogenous pseudocyclic electron flow, detected as a Mehler reaction, was also considerable (100 to 150 μeq/hr · mg Chl with methylamine). Diaminodurene supported a high rate of photosystem I-mediated electron flow to methylviologen (400 to 750 μeq/hr · mg Chl).     

Serum tolbutamide and chlorpropamide concentrations in patients with diabetes mellitus

A selective and sensitive gas chromatographic technique was used to measure the steady-state serum concentrations of tolbutamide and chlorpropamide in 97 patients with maturity-onset diabetes mellitus who had been taking these drugs (37 tolbutamide, 60 chlorpropamide) for at least a year. No other antidiabetic agents had been given. The serum tolbutamide concentrations varied widely between the patients (from close to zero to 370 μmol/l (100 μg/ml)), yet the variation in dosage was only sixfold (0·5-3·9 g daily). The serum chlorpropamide concentrations varied even more widely (from close to zero to 882 μmol/l (244 μg/ml)), though the dosage variation was fourfold (125-500 mg daily). There was no systematic relation between dosage and serum concentrations of the drugs.Only 2 (5·4%) of the tolbutamide-treated patients and 10 (16·7%) of the chlorpropamide-treated patients had normal fasting blood glucose concentrations (below 5·5 mmol/l (99 mg/100 ml)), and fewer than half had values below 8·0 mmol/l (144 mg/100 ml). In most cases, therefore, the treatment was insufficient.There was no significant difference in mean fasting blood glucose concentrations between the two treatment groups. The mean steady-state concentration of chlorpropamide, however, was significantly higher than that of tolbutamide. Thus, contrary to common belief, the intrinsic activity of chlorpropamide is apparently not greater than that of tolbutamide. The alleged greater potency of chlorpropamide seems to be related wholly to kinetic differences, such as the less extensive metabolic degradation and slower elimination of the drug.We conclude that treatment with sulphonylureas in conventional dosage is far from optimal and that monitoring the concentrations of these drugs in the blood may help to improve their efficacy.     

Detection of Phosphatidylcholine-Coated Gold Nanoparticles in Orthotopic Pancreatic Adenocarcinoma using Hyperspectral Imaging

Nanoparticle uptake and distribution to solid tumors are limited by reticuloendothelial system systemic filtering and transport limitations induced by irregular intra-tumoral vascularization. Although vascular enhanced permeability and retention can aid targeting, high interstitial fluid pressure and dense extracellular matrix may hinder local penetration. Extravascular diffusivity depends upon nanoparticle size, surface modifications, and tissue vascularization. Gold nanoparticles functionalized with biologically-compatible layers may achieve improved uptake and distribution while enabling cytotoxicity through synergistic combination of chemotherapy and thermal ablation. Evaluation of nanoparticle uptake in vivo remains difficult, as detection methods are limited. We employ hyperspectral imaging of histology sections to analyze uptake and distribution of phosphatidylcholine-coated citrate gold nanoparticles (CGN) and silica-gold nanoshells (SGN) after tail-vein injection in mice bearing orthotopic pancreatic adenocarcinoma. For CGN, the liver and tumor showed 26.5±8.2 and 23.3±4.1 particles/100μm² within 10μm from the nearest source and few nanoparticles beyond 50μm, respectively. The spleen had 35.5±9.3 particles/100μm² within 10μm with penetration also limited to 50μm. For SGN, the liver showed 31.1±4.1 particles/100μm² within 10μm of the nearest source with penetration hindered beyond 30μm. The spleen and tumor showed uptake of 22.1±6.2 and 15.8±6.1 particles/100μm² within 10μm, respectively, with penetration similarly hindered. CGH average concentration (nanoparticles/μm²) was 1.09±0.14 in the liver, 0.74±0.12 in the spleen, and 0.43±0.07 in the tumor. SGN average concentration (nanoparticles/μm²) was 0.43±0.07 in the liver, 0.30±0.06 in the spleen, and 0.20±0.04 in the tumor. Hyperspectral imaging of histology sections enables analysis of phosphatidylcholine-coated gold-based nanoparticles in pancreatic tumors with the goal to improve nanotherapeutic efficacy.     

Schlemm’s Canal and Trabecular Meshwork in Eyes with Primary Open Angle Glaucoma: A Comparative Study Using High-Frequency Ultrasound Biomicroscopy

We investigated in vivo changes in Schlemm’s canal and the trabecular meshwork in eyes with primary open angle glaucoma (POAG). Relationships between Schlemm’s canal diameter, trabecular meshwork thickness, and intraocular pressure (IOP) were examined. Forty POAG patients and 40 normal individuals underwent 80-MHz Ultrasound Biomicroscopy examinations. The Schlemm’s canal and trabecular meshwork were imaged in superior, inferior, nasal and temporal regions. Normal individuals had an observable Schlemm’s canal in 80.3% of sections, a meridional canal diameter of 233.0±34.5 μm, a coronal diameter of 44.5±12.6 μm and a trabecular meshwork thickness of 103.9±11.1 μm, in POAG patients, Schlemm’s canal was observable in 53.1% of sections, a meridional canal diameter of 195.6±31.3 μm, a coronal diameter of 35.7±8.0 μm, and a trabecular meshwork thickness of 88.3±13.2 μm, which significantly differed from normal (both p <0.001). Coronal canal diameter (r = -0.623, p < 0.001) and trabecular meshwork thickness (r = -0.663, p < 0.001) were negatively correlated with IOP, but meridional canal diameter was not (r = -0.160, p = 0.156). Schlemm’s canal was observable in 50.5% and 56.6% of POAG patients with normal (<21 mmHg) and elevated (>21 mmHg) IOP, respectively (χ = 1.159, p = 0.282). Coronal canal diameter was significantly lower in the elevated IOP group (32.6±4.9 μm) than in the normal IOP group (35.7±8.0 μm, p < 0.001). This was also true of trabecular meshwork thickness (81.9±10.0 μm vs. 97.1±12.0 μm, p < 0.001). In conclusion, eyes with POAG had fewer sections with an observable Schlemm’s canal. Canal diameter and trabecular meshwork thickness were also lower than normal in POAG patients. Schlemm’s canal coronal diameter and trabecular meshwork thickness were negatively correlated with IOP.     

Ethylene, a regulator of young fruit abscission

In an earlier study we reported that detached cotton flowers produced sufficient ethylene before the period of natural abscission to suggest that ethylene might be a natural regulator of young fruit abscission. The present report explores this probability further. Intact cotton (Gossypium hirsutum L.) fruits produced ethylene at rates as high as 36 μl ethylene/kg fresh wt·hr during the 2 days before they abscised. Direct measurements of ethylene in gas samples withdrawn from fruits indicated that production of 1 μl ethylene/kg fresh wt·hr is equivalent to an internal concentration of approximately 0.1 μl/l. Fumigation of fruiting cotton plants with only 0.5 μl/l caused 100% abscission of young fruits and floral buds within 2 days. This correlated with the estimated endogenous levels of ethylene. Reduced pressure, which reduced the internal levels of ethylene, delayed abscission of young fruits and leaves, a result which supports our conclusion from this study— that ethylene is one of the regulators of young fruit abscission in cotton.     

Spirulina platensis extract addition to semen extender enhances cryotolerance and fertilizing potentials of buffalo bull spermatozoa

The objective of this study was to investigate the effect of Spirulina platensis extract (SPE) addition to the freezing extender on freezability, lipid peroxidation, ultrastructure alterations and fertilizing potentials of frozen-thawed buffalo bull spermatozoa. Semen samples were collected with artificial vagina from five adult fertile bulls and diluted with Tris-base extender containing SPE (1, 5, 10 and 20 μg/mL) or without SPE (control). Diluted semen was cooled to 4 °C throughout one hour and frozen in 0.25 mL straws: prior to being stored in liquid nitrogen. Cryopresreved spermatozoa were assessed for post-thawing sperm motility, viability, acrosomal integrity, ultrastructure changes, antioxidant activities, lipid peroxidation and fertility rate. The current results clearly indicated that adding 10μg/mL SPE to the freezing extender significantly improved (P< 0.05) post-thawing motility and decrease the percentage of acrosomal damage (51.67±6.02% and 16.33±1.46%, respectively) compared with the control (28.33±4.41% and 26.33±1.77%, respectively). Moreover, addition of 10 μg/mL SPE to the semen extender significantly diminished (P< 0.05) MDA concentration (10.66±2.40 nmol/10⁹) compared with the control (22.66±4.26 nmol/10⁹). Therefore, the present results revealed that addition of 10μgl/mL SPE to the freezing extender might improve semen quality and reduce cryodamage of the buffalo bull spermatozoa.